A major disadvantage with traditional tree breeding, especially for forest tree species, is the slow progress due to their long generation periods. However, by taking advantage of recent developments in gene technology the time required to produce a new variety could be reduced significantly. In addition, a biotechnological approach would allow closer targeting of traits considered desirable by the forest and pulp industries, in specific tree species.
To date, most applications of genetic engineering of trees have focused on modifying lignin biosynthesis, resulting in trees with less lignin or a modified lignin composition, earlier flowering, pest or herbicide resistance. In order to change growth and development processes in trees, the manipulation of plant hormone levels, or the hormone sensitivity, would also be of interest. However, as yet there have only been few examples of the modification of plant hormone levels in trees. These have mainly been accomplished by directly altering endogenous IAA biosynthesis or cytokinin biosynthesis or indirectly by modifying various hormone pools using the Agrobacterium rolC gene. Although such modifications in all cases lead to trees with altered growth characteristics and wood properties, so far no improvements with a clear practical application have been obtained.
Gibberellins (GAs) are a group of more than 100 tetracyclic diterpenes, some of which are essential endogenous regulators that influence growth and development processes throughout the plant life cycle, e.g. shoot elongation, the expansion and shape of leaves, flowering and seed germination. The best examples illustrating the importance of GAs in control of shoot elongation are GA-deficient mutants of Arabidopsis, maize and pea. These have reduced levels of active GA(s) compared to wild type plants, resulting in a dwarfed phenotype due to a reduction in internode length. The phenotype of such GA-deficient mutants can be completely restored by the application of an active GA. At the cellular level, GAs have been found to promote both cell division and cell elongation.
Biosynthesis of GAs in planta occurs through the isoprenoid pathway from mevalonic acid. Gibberellin levels are mainly regulated by transcriptional control of gibberellin biosynthesis genes. In particular, the multifunctional enzyme gibberellin 20-oxidase (GA 20-ox) is a key-enzyme in controlling GA biosynthesis (FIG. 1). It catalyses the stepwise conversion of the C-20 gibberellins, GA12/GA53, by three successive oxidations to GA9/GA20, which are the immediate precursors of the active gibberellins, GA4 and GA1, respectively. The expression of the GA 20-oxidase gene is down regulated by the action of GA1/4, suggesting that direct end-product repression is involved in regulation of the gene. In addition, some authors have suggested that GA 20-oxidase is photoperiodically regulated at the transcription level.
Application of chemicals that alter GA levels in the plant is a common practise in traditional agriculture and horticulture. Inhibitors of GA biosynthesis are especially commonly used as growth retardants in cereals and ornamental plants. In order to reduce the use of these chemicals, a biological approach like genetic modification of endogenous GA biosynthesis would have clear advantages. Using Arabidopsis as a model organism it has been shown that it is possible to change GA levels by modifying GA 20-oxidase enzyme levels and that this results in plants with altered growth and development patterns. Transgenic Arabidopsis expressing the GA 20-oxidase in a sense orientation shows earlier flowering and taller stems than wild type plants, whereas antisense plants have the reverse properties [Coles, J. P. et al. Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes. Plant J. 17, 547-556 (1999)].
Modification of GA biosynthesis in a higher species, such as trees would be of additional interest since this would open up ways to modify wood. Previous hormone application studies have shown that GAs are required for the differentiation of xylem fibres, and that they have pronounced effects on the length of secondary xylem fibres and on both longitudinal and radial growth in hard wood species and conifers.
Obviously there remains a need for improved methods for modification of the growth properties of trees, in particular properties of technical and economical interest, such as growth rate, biomass increase and fibre length. Likewise, there remains a need of transgenic trees, exhibiting improved properties, such as increased growth rate, stem volume and xylem fibre length. Consequently, the objective of the present invention is to provide such improved methods and transgenic trees. Another objective is to reduce or eliminate the use of growth influencing chemicals in forestry.